Transforming growth factor-beta (TGF-beta) has dual and paradoxical functions as a tumor suppressor and promoter of tumor progression and metastasis. In spite of absence of mutations in TGF-beta receptors, melanoma cells are resistant to its growth inhibitory activity. Furthermore, melanoma cells increase production and secretion of TGF-beta, which upon activation induces matrix deposition, angiogenesis, survival and transition to more aggressive phenotypes. We have demonstrated that SKI functions as a molecular switch, converting the TGF-beta co-regulator proteins Smad2/3 from an activating to a repressing entity on chromatin. SKI is also a potent activator of the Wnt signaling pathway, targeting MITF and NrCAM, two genes involved in survival, migration and invasion. Based on these findings and new preliminary data we hypothesize that SKI functions as a sensor and modifier of environmental signals, protecting melanoma cells from growth inhibition and apoptosis and stimulating proliferation and invasion. To test this hypothesis, we will: Aim 1: Determine the molecular mechanism(s) by which SKI senses and modifies growth inhibitory signals. We will use endogenous and exogenous SKI to determine how SKI complexes respond to inhibitory and/or apoptotic signals mediated by TGF-beta. Analysis and characterization of SKI protein complexes will be performed using new HPLC fractionation/co-immunoprecipitations techniques and Mass Spectrometry. Functional relevance of SKI complexes will be validated by promoter-reporter assays, sequential chromatin Immunoprecipitations, and gel-shift assays. Aim 2: Determine whether knocking down SKI prevents melanoma growth and metastasis in a mouse xenograft model. We will use proven SKI RNAi technology to test melanoma tumor formation using xenografts containing human stroma-derived fibroblasts. Aim 3: Test whether SKI cooperates with the RAS signaling pathway to induce melanoma progression in vivo. We will analyze in detail molecular requirements needed for switching SKI from a tumor suppressor to a tumor promoter using a syngeneic mouse model. Together, the three aims will likely identify and characterize a cascade of molecular and cellular events regulated by SKI that can trigger cell cycle aberrations, increased tumorigenicity and metastatic potential of melanocytes.